[Micro] growth and culturing of bacteria

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings
Growth & Culturing
of Bacteria
Microbiology 130
Chapter 6

 Microbial Growth Defined:
 Microbial growth is the increase in number of cells,
not cell size
 Mother cells
 Daughter cells
 Cell Division:
 Binary fission
 Tetrads
 Sarcinae
 Budding
Microbial Growth and Cell Division

Reproduction in Prokaryotes
 Binary fission
 Budding
 Conidiospores (actinomycetes)
 Fragmentation of filaments

Binary Fission

Phases of Growth
 4 Phases:
 1) Lag phase-
 2) Log (logarithmic) phase
 3) Stationary phase
 4) Decline phase or death phase

Log Phase

Log Phase with Calculations
 If 100 cells growing for 5 hours produced 1,720,320
cells:

Log phase

4 Phases of Microbial Growth

Growth in Colonies
 A pure culture contains only one species or strain.
 A colony is a population of cells arising from a single
cell or spore or from a group of attached cells.
 A colony is often called a colony-forming unit (CFU).

Measuring Bacterial Growth
Serial Dilutions
 Direct Measurements of Microbial Growth
 Plate counts: Perform serial dilutions of a sample

Standard Plate Count
Inoculate
Petri plates
from serial
dilutions
2 methods:
Pour Plate
Spread Plate

Plate Count
After incubation, count
colonies on plates that have
25-250 colonies (CFUs)

Direct Measurements of Microbial Growth
Filtration

 Multiple tube
MPN test.
 Count positive
tubes and
compare to
statistical
MPN table.

Direct microscopic count

Most Probable Number
 Estimate of number
 MPN 5 test tubes 10, 1, and 0.1 ml
 Growth is displayed by production of gas bubbles or by
becoming cloudy
 Estimates are from a known chart ( table 6.1 pg 149)

Other Methods: Estimating Bacterial Numbers
by Indirect Methods
Turbidity

Measuring Microbial Growth
Direct methods
 Plate counts
 Filtration
 MPN
 Direct microscopic count
 Dry weight
Indirect methods
 Turbidity
 Metabolic activity
 Dry weight

Factors Affecting Bacterial Growth
The Requirements for Growth:
Physical Requirements
 Temperature
 Minimum growth temperature
 Optimum growth temperature
 Maximum growth temperature

Temperature
Figure 6.1

Psychrotrophs/Psychrophiles
 Grow between 0°C and 20-30°C
 Cause food spoilage

Psychrotrophs/Psychrophiles
Figure 6.2

 pH
 Most bacteria grow between pH 6.5 and 7.5
 Molds and yeasts grow between pH 5 and 6
 Acidophiles grow in acidic environments

Mesophiles & Thermophiles
 Mesophiles-
 grow best between 25 degrees C and 40 degrees C
 Thermophiles-
 Heat loving- grow best at 50 - 60 degrees C
 Obligate thermophiles
 Facultative thermophiles-

Physical Factors - Chemical Requirements
Oxygen (O2)

Toxic Forms of Oxygen
 Singlet oxygen: O2 boosted to a higher-energy state
 Superoxide free radicals: O2
–
 Peroxide anion: O2
2–
 Hydroxyl radical (OH•)

Physical Factors / Requirements
 Osmotic pressure
 Hypertonic environments, increase salt or sugar,
cause plasmolysis
 Extreme or obligate halophiles require high osmotic
pressure
 Facultative halophiles tolerate high osmotic pressure

Figure 6.4

The Physical Requirements for Growth
 Moisture-
 Hydrostatic Pressure-
 Radiation-

Nutritional Factors - Chemical Requirements
 Carbon
 Structural organic molecules, energy source
 Chemoheterotrophs use organic carbon sources
 Autotrophs use CO2

 Nitrogen
 In amino acids and proteins
 Most bacteria decompose proteins
 Some bacteria use NH4
+
or NO3
–
 A few bacteria use N2 in nitrogen fixation
 Sulfur
 In amino acids, thiamine and biotin
 Most bacteria decompose proteins
 Some bacteria use SO4
2–
or H2S
 Phosphorus
 In DNA, RNA, ATP, and membranes
 PO4
3–
is a source of phosphorus

 Trace elements
 Inorganic elements required in small amounts
 Usually as enzyme cofactors
 Vitamins- organic substances and growth factors
 Organic compounds obtained from the environment
 Vitamins, amino acids, purines, and pyrimidines
 Nutritional Complexity
 Locations of Enzymes
 Adaptations to Limited Nutrients

Sporulation / Endospores
 Formation of endospores in Bacillus, Clostridium
 and G+ genera
 Can Survive long periods of drought
 Axial nucleoid
 Endospore septum grows
 Spore coat and Exosporium
 Germination- 3 stages:
 1) Activation, 2) germination, 3) outgrowth

Culturing Bacteria
 Methods of Obtaining Pure Culture
 1) The Streak Plate Method – sterile wire loop and
streaked in different patterns on agar
 2) Pour Plate Method- serial dilutions using melted
agar

Streak Plate

Culture Media
 Culture medium: Nutrients prepared for microbial
growth
 Sterile: No living microbes
 Inoculum: Introduction of microbes into medium
 Culture: Microbes growing in/on culture medium
 Synthetic media
 Defined synthetic media
 Complex media

Agar
 Complex polysaccharide
 Used as solidifying agent for culture media in Petri
plates, slants, and deeps
 Generally not metabolized by microbes
 Liquefies at 100°C
 Solidifies ~40°C

Culture Media
 Chemically defined media: Exact chemical composition
is known
 Complex media: Extracts and digests of yeasts, meat,
or plants
 Nutrient broth
 Nutrient agar

Culture Media
Tables 6.2, 6.4

Anaerobic Culture Methods
 Reducing media
 Contain chemicals (thioglycollate or oxyrase) that
combine O2
 Heated to drive off O2

 Anaerobic
jar
Figure 6.5

 Anaerobic
chamber
Figure 6.6

Capnophiles Require High CO2
 Candle jar
 CO2-packet
Figure 6.7

Selective Media
 Suppress unwanted
microbes and
encourage desired
microbes.
Figure 6.9b–c

Differential Media
 Make it easy to distinguish colonies of different
microbes.
Figure 6.9a

Enrichment Media
 Encourages growth of desired microbe
 Assume a soil sample contains a few phenol-degrading
bacteria and thousands of other bacteria
 Inoculate phenol-containing culture medium with the
soil and incubate
 Transfer 1 ml to another flask of the phenol medium
and incubate
 Transfer 1 ml to another flask of the phenol medium
and incubate
 Only phenol-metabolizing bacteria will be growing

Preserving Bacteria Cultures
 Deep-freezing: –50°to –95°C
 Lyophilization (freeze-drying): Frozen (–54° to –72°C)
and dehydrated in a vacuum

Methods of Performing Multiple
Diagnostic Tests
 Enterotube Multitest API- simultaneous testing
 To identify enteric bacteria- cause food poisoning,
typhoid, shigellosis, gastroenteritis etc
 Living, But Non-culturable organisms:
 Some microbs can be observed with microscope,
identify their DNA but they can not be cultured

[Micro] growth and culturing of bacteria

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